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Detection and Quantification of -2-Microglobulin Using

Mass Spectrometric Immunoassay

Kemmons A. Tubbs, Dobrin Nedelkov, and Randall W. Nelson 1

n tr in sic B ioprobes, I nc., 625 South Smi th Road, Sui te 22, T empe, Ar izona 85281

eceived J une 20, 2000

The use of mass spectrometric immunoassay

MSIA) in analyzing-2-microglobulin (2m) present

n human biological fluids (tears, saliva, plasma, andrine) is described. Pipettor tips containing porous

ffinity frits, derivatized with polyclonal anti-2m

mmunoglobulin, were manufactured and used to se-

ectively isolate and concentrate 2m from the

biofluids, after which matrix-assisted laser desorp-

ion/ionization time-of-flight mass spectrometry was

sed to detect 2m unambiguously at its character-

stic molecular mass. The affinity tips were found

apid to use, requiri ng approximately 15 min per

nalysis, and exhibited low nonspecific binding

properties that yielded essentially interference-free

nalyses. The 2m MSIA was made quantitative bynclusion of an internal standard into the analysis

or signal normalization. T he resulting assay had a

inear dynamic range (R 2 0.983) coveri ng a 2m

oncentration range of 0.010–1.0 mg/L with a stan-

ard error of approximately 5%. In application,

rine samples from healthy individuals were

creened and compared with sample from an indi-

idual sufferi ng from renal infection. Results i ndi-

ated an approximately 30-fold increase in 2m lev-

ls in samples taken from the infected individual.

During the screening, MSIA was able to distinguish

between wild-type and glycosylated forms of 2m,which made possible the accurate quantification of

wild-type 2m without interference from glycosy-

ated versions of the protein. These results demon-

trate a new approach to the rapid and accurate

etection/quantification of 2m present in biological

uids. © 2001 Academic Press

-2-Microglobulin (2m)2 is a low-molecular-ma s

protein identifi ed as th e light cha in of the Clas s I ma johistocompat ibil ity complex synth esized in a l l n ucle

ated cells. Upon activation of the immune system, botB- and T-lymphocytes actively release 2m into circul at i o n whe r e i t i s l a t e r e l i mi nat e d v i a g l o me r u l ar fi ltra tion a nd t ubular rea bsorption. S erum levels of 2mhav e b ee n me as u r e d a nd nomi nal ly cor r e lat e d t o a iments such as AIDS (1, 2), rheumatoid arthrit is (3)leukemia (4), myeloma (5), a nd ma ligna nt lymphom(6). On the other ha nd, 2m levels in urine ar e indicators of glomerular fi l tra tion rat e and t ubula r reabsorption (7). Conventiona lly, 2m levels a re monitored usin g a v a r iet y of im m un o-b a s ed a s s a y s, in clu dinenzyme-linked immunosorbent a ssa ys (8), ra dioimmu

noassays (9), and particle-enhanced turbidimetry assays (10). The quanti tative dynamic range (spannin2m concentra tions of 0.2–20 mg/L) a nd t he a ccura c(1–10%) of t hese a ssa ys a re suffi cient to cover th e norma l an d eleva ted levels of 2m in a va riety of biologicafluids. One drawback of the conventional assays, however, is the inabil i ty to dif ferentiate between the wilt y p e an d v ar i ant s of 2m. Such distinction is import a nwhe n co ns i d e r i ng t hat g e ne t i c and p o s t t r ans l at i o nav a r i a n t s o f 2m ar e i nd i cat i v e o f a i l me nt s d i f f e r e nfrom those indicat ed by the wild-ty pe protein. Sepa ra tassays using monoclonal antibodies are thus requireto discern betw een t hese different protein forms, a nd tdate, no such combination of assays has been used ithe study of 2m .

1 To whom correspondence should be addressed. Fax: (480) 804-

778. E-mail: [email protected].

2 Abbreviations used: 2m , -2-microglobulin; MALDI-TOF MS

ma tr ix-a ssisted la ser desorption/ioniza tion tim e-of-fl ight m as s spet romet ry; M SI A , ma s s s pect romet ric immunoa s s a y; I gG, immuno

globulin; CMD, carboxylated dextran; EDC, 1-ethyl-3-(3-dimethyam inopropyl)carbodiimide; ddH 2O , d ou bl y d is t il le d w a t e r ; H B

buffer, 10 mM H epes, pH 7.4, 0.15 M NaC l, 0.005%S urfa ctant P 2

E2m, equine 2m; H2m, huma n 2m; SD S, sodium dodecyl sulfat

6 0003-2697 /01 $35.0Copyright © 2001 by Academ ic Pre

All r ights of reproduction in any form reserve

nalytical Biochemistry 289, 26–35 (2001)

oi:10.1006/a bio.2000.4921, a va ila ble online a t ht tp://w ww .idea libra ry.com on

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Two protein ma ss spectrometry techniques, ma trix-ssist ed la ser desorpt ion/ioniza t ion tim e-of-fl ight m a sspectr ometry (MALD I-TOF MS) (11–13) a nd electro-pray ionization mass spectrometry (14), offer the par-cular advantage of dif ferentiating between dif ferent

mas s -s hi ft e d f o rms of t he s a me p r ot e in. I n t hi s ma n-e r , a s i ng l e p anant i b o d y can b e u s e d t o r e t r i e v e a l lrotein forms from a biological fluid, upon which each

orm is detected during ma ss spectrometry a t a uniquend characteristic molecular mass. Resolution of re-at e d s p e ci e s a l s o a l l o ws mas s -s hi f t e d v ar i ant s o f aa rget protein to be intentionally incorpora ted int o then a l ys is f or u s e a s i nt e r na l r ef er en ce s t a n d a r d s f oru ant i t a t i v e anal y s i s . T hi s s t e p f o r ms t he b as i s o f a

mas s s pe ct r o met r i c i mmu noas s a y (MS I A), an as s a yhat can b e u s e d f o r t he u namb i g u o u s d e t e ct i o n andigorous quantification of polypeptides retrieved fromomplex b iologica l s yst ems (15).

This report focuses on the use of MSIA in detectingnd q u ant i f y i ng 2m present in several biologica l fl u-

ds. The general MSIA approach follows our previouslyeported methodology (15, 16) and is shown in Fig. 1.ipettor t ips (termed MSIA tips), containing porousolid supports covalently derivatized with polyclonaln t i -2m i mmu no g l o b u l i n (I g G ), ar e u s e d t o e x t r act

2m (and i ts variants) from biological samples by re-eatedly flowing the samples through the t ips. A sec-n d 2m species (mass-shif ted va riant of 2m dopednto the samples at a constant concentration) is coex-ra cted w ith th e indigenous 2m and i s u se d as a q u an-t at i ve i nt er na l r e fe r ence s t an d ar d . N ons p eci fi cal lyound compounds a re rinsed from the t ip using a series

f b u f f e r and wat e r r i ns e s , a f t e r whi ch t he wi l d -t y p e2m, 2m v a r ia n t s , a n d t h e in t er n a l r ef er en ce a r elu t e d f r om t he MS I A t i ps d ir e ct l y ont o a t a r g et i nreparation for MALDI-TOF. Mass spectrometry then

ollows with the retained proteins identified via accu-ate molecular mass determination. 2m levels are de-e r mine d v ia a q u an t i t a t i ve me t hod i n w hi ch t he 2mi g n a l s a r e n o r m a l i z e d t o t h e s i g n a l o f t h e i n t e r n a le f e r e nce and t he v al u e s ar e co mp ar e d t o a wo r k i ngurve constructed from sa mples conta ining know n con-entra tions of 2m.

G i v en h er e a r e r es u lt s d em on s t r a t i n g t h e u se of

MSIA in detecting 2m in four biological fluids (tears,las ma , s a l iv a , and u r i ne) and i n r i g or ou s ly q u ant i fy -

n g 2m present in human urine samples. The objec-ves of investigation were: (i) to construct MSIA t ips

ha t e xhi bi t a l ow d eg r ee o f no ns pe ci fi c b i nd ing an dvaluate the tips by screening the biological fluids; (ii)o d es ig n a n a s s a y w i t h a n a d e q ua t e q u a n t i t a t i ve d y -amic range, accuracy, and l inearity to cover the con-entra tions of 2m expected in t he biofluids; (iii) to use

M S I A i n a l im i t ed q u a n t i t a t i v e e va l u a t i on of u r in eamples obtained from healthy individuals and an in-

dividual with renal dysfunction; and (iv) to use MSIAin the simultaneous detection of wild-type and posttranslationally modified 2m present in urine.

MATERIALS AND METHODS

M S I A T i p M a n u fa ct u r i n g

Frits for the prototype MSI A tips (Int rinsic B ioprobeInc., Tempe, AZ) were m a nufa ctured to t he specifi cationof commercially available wide-bore P-200 pipettor tip

FIG. 1. I l lus t ra t ion of t he M SI A procedure. A na lyt es a re s electively retrieved from solution by repetitive flow through a receptoderiva t ized M SI A t ip. Once w a s hed of t he nons pecifica lly boun

compounds, the reta ined species are eluted onto a ma ss spectrometet a r get us ing a M AL DI ma t rix. M AL DI -TOF M S t hen follow s , w it

analytes detected at precise m /z va lues . The a na lys es a r e qua li t a t iv

b y n a t u r e b u t ca n b e m a d e q u a n t i t a t i v e b y i n cor p or a t i n g m a ss hif t ed va ria nt s of t he a na lyt e int o t he procedure for us e a s int erna

s t a n d a r d s .

2-2-M I CR OGL OB U L I N M ASS SP ECTR OM ETR I C I M M U NOASS AY

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Coast Scientifi c, Sa n Diego, C A, P T 0340-B YN) by usingnnealing molds made of stainless steel (type 446; 100–50 holes of 0.071 in. (entrance) per mold; 2° taper, pol-

s hed and t r eat e d wi t h g r aphit e r el eas e ag ent ). Themolds were loaded with soda lime glass spherical beads150–200 m; 75% SiO2, 15% Na 2O , and 10% C aO ;igma, St. Louis, MO), and annealing was achieved in anrgon-backfilled furnace by ramping the temperat ure

rom 772°C (equilibrat ed, t 0) to 800°C (t 3 min;quilibrate for 1 min). Upon completion of the ramp an-eal the molds w ere immedia tely removed from t he ovennd the frits were taken out. The process yielded frits

with high-fl ow cha ra cteristics a nd appropria te bore anda per to fi t the entran ce of t he wide-bore P -200 pipetteps (room temperat ure frit dimensions, 0.061 in. (en-

rance), 0.092 in. (length), 2° taper).The fri ts were activated and derivatized in batches

30–50 per batch) prior to packing into the pipettorps. After acid conditioning (with 0.05 M HCl for 1 h,ir-dried), the frits w ere treat ed wit h 10%am inopropyl

r iethoxysila ne (Aldrich, Milw a ukee, WI) in a nhy drouso lu ene f or 12 h a t r oom t e mpe r at u r e . The am i ne-unctionalized fr i ts were t hen equil ibrat ed in reactionuffer (100 mM sodium phosphate, pH 4.8, 100 mM

N aC l ) f or 15 mi n i n a r e act i on v es s el u nd e r s li g htacuum. After equil ibration, the buffer was replaced

with a mixture of 15-kDa molecular mass carboxylatedextra n (CMD, Fluka, Milwa ukee, WI) a nd 1-ethyl-3-

3-dimethylaminopropyl)ca rbodiimide (ED C, Sigma )10 mg/mL each in t he rea ction buffer) a nd t he a ir w a sgain evacuated from the reaction vessel. The reaction

was al lowed to proceed for 1 h (with two subsequent

dditions of ED C t o the rea ction mixture at 20 a nd 40min int o the rea ction) before termina ting a nd r insing.

rior to coupling of the antibody, the CMD-amplifiedrits w ere rinsed vigorously wit h 100 mM sodium phos-hat e , p H 8.0, 0 .5 M N a C l . The f r i t s w e r e t he n a ct i -a t ed for 10 m in w i th E D C /N -hy droxysu ccinim ide

S igma ) (100 mM ea ch, in H 2O) a nd incubat ed with t hef fi ni t y -p u r i fi e d r ab b i t ant i -hu man 2m I g G (D AK O ,a rpinteria, CA) (0.1 m g/mL, in 20 mM sodium a ce-

a t e , p H 4.7). U ncou pl ed ant i bod y wa s r e mov ed b yxtensive rinsing w ith HB S buffer (10 mM Hepes, pH.4, 0.15 M Na Cl, 0.005% Surfa cta nt P 20). This m a n-

facturing process yielded MSIA tips with a bindingapa city est ima ted a t 10-100 pmol, w hile ha ving a dea dolume of approximately 1.5 L. The anti-2m M S I Aps w ere found t o be sta ble and a ctive for a period of a t

ea st 3 months following a ntibody immobilization (byt or i ng a t 4° C i n H B S b u ff er ).

B i oflu i d s

A l l flu i d s we r e o b t ai ne d i mme d i at e l y p r i o r t o u s e ;r ot e as e i nhib it o r cock t ai l (s et I I I , C a l bi oche m, La

J olla, C A) w a s a dded immediat ely in order t o minimizpossible proteolytic degra da tion of 2m .

Tears. Hum a n tears were collected by wa shing theye with doubly distilled water (ddH 2O) an d collectinthe rinse. A 20-L aliquot of the eye rinse was mixewith 180 L HBS buffer and used as stock tears solution. This stock was further diluted by a factor of 1with either water (for MALDI-TOF analysis) or HB

buffer (for MSIA analysis).Plasma. A tota l of 44.7 L of human whole bloo

wa s collected under steri le conditions from a lancep unct u r e d fi ng er u s ing a he par i ni z ed mi cr ocol u m(Drumm ond Scientifi c Co., B rooma ll, P A), m ixed w it205 L H B S buffer, an d cent rifuged for 30 s (a t 7000 gto pellet the red blood cells. A 50-L a l i q u o t o f t hs up er n a t a n t w a s m ix ed w i t h 200 L H B S a n d t hresulting solution was used for MSIA; an al iquot wafurther diluted (10 fold) with ddH 2O for MALDI-TOanal y s i s .

Sa l iv a . Huma n whole sa l iva wa s diluted by a facto

of 100 i n d d H 2O or H B S b uf fer i n pr ep a r a t i on f oMALDI-TOF or MSIA, respectively.

U r in e . H u m a n u rin e w a s pr epa r ed for M AL DTOF by a 100-fold dilution with ddH 2O; a 2-fold dilution with HBS buffer was used for MSIA.

M S I A

MSIA w a s performed on t he biofl uids by repea tedld r aw i ng a fl u i d (20 times) thr ough a n a nti-2m MS I Atip using a ha nd-held P -200 m icropipettor. After thr e p e t i t i v e -flo w i ncu b at i o n, t he t i p was r i ns e d wi t h

m L o f H B S b u f f e r ( b y d r a w i n g t h e H B S t h r o u g h i200-L al i q u o t s and t he n d i s car d i ng ), f o l l o we d b y 1 -mL r i ns e wi t h d d H 2O ( u s i n g t h e s a m e w a s h a nd is ca r d a p pr oa c h ). At t h e fi n a l d is ca r d of t h e w a t er i ns e , i t was che ck e d t hat a l l r e s i d u al wat e r was e xp el le d f r om t he t i p. The r e t ai ned comp ou nd s we reluted from the tip by dra w ing a 3-L a liquot of mat risolution (saturated solution of -cyano-4-hydroxycinnamic acid (Aldrich) in 1:2, acetonitrile:ddH 2O, 0.2%trifluoroacetic acid) into the t ip (enough to cover thfrit), upon w hich t he ma tr ix/eluent mix w a s depositedirectly onto a MALDI-TOF target. MALDI-TOF M

w a s per f or m ed u si n g a m a s s s pe ct r om et e r t h a t h abeen described previously (17). B riefly , t he inst rumenuses a two-stage 30-kV (2 1 cm; 15 kV/st a ge) cont inu ou s -e xt r a ct i on s ou r ce t o acce le r at e i ons t o t he e nt r a nce of a 1.4-m fl i ght t u b e cont a i ning an i on g u idwire. Ions genera ted using a pulsed N 2 la ser (337 nmw ere detected using a hybrid single-chan nel plat e/discreet-dynode m ultiplier biased a t 3.8 kV. Spectrwe r e r e cor d ed u s ing an av e r ag i ng t r a ns i ent r e cor d ewhile monitoring individual laser shots using a separ at e o s ci l l o s co p e and at t e nu at i ng l as e r i nt e ns i t y ( i

8 TU B B S , N E D E L K O V , AN D N E L S O N

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ea l time) during a cquisition. All spectr a w ere a cquiredn the positive-ion mode.

Quantification

Internal reference. E q u i ne 2m (E2m) wa s chosens an i nt er nal r e fe r ence f or q u ant i fi cat i on b ecau s e oft s h i g h d e g r e e o f s i m i l a r i t y t o h u m a n 2m (H2m )75%sequence homology) a nd resolva ble ma ss d iffer-n ce f rom H2m (M WE2m 11,402.9; MWH2m 1,729.7) an d because i t wa s easily obtaina ble. H orserine was collected fresh (at a local stable) and treated

mmediately with protease inhibitor cocktail. Low sol-b i l i t y co mp o u nd s we r e r e mo v e d f r o m t he u r i ne b yvernight refrigeration (at 4°C) followed by centrifuga-on f or 5 m i n a t 5 0 0 0g . The urine was then concen-

r a t e d 20-f ol d ov er a 10-k D a MW cu t of f fi l t er , wi t hepeti t ive HB S a nd wa ter r inses and w ith several fil t erxcha nges (four fi lters/200 m L urine). Trea tm ent of00 mL fresh urine resulted in 10 mL of 2m-enriched

orse urine which served as stock internal referenceolution for 100 a na lyses.

Working curve. Q ua n t i fi ca t i on of H2m w a s p e r -ormed following protocols that have been outlined pre-iously (15, 16). Briefly, standards were prepared bytepwise dilution (i.e., 0.8, 0.6, 0.4, 0.2, and 0.1, in

HB S) of a 1.0 mg/L stock H2m solution to a concen-r a tion of 0.1 mg/L; th e 0.1 mg/L solution served a stock for an identical stepwise dilution covering theecond deca de in concentra tion (0.01– 0.1 mg/L). Alank solution containing no H2m w as a l so pr e par e d .

The sa mples for MS IA were prepar ed by mixing 100 L

f ea c h of t h e s t a n d a r d s w i t h 100 L of stock horserine a nd 200 L of HB S buffer. MSIA wa s performedn e ach s amp l e as d e s cr i b e d ab o v e , r e s u l t i ng i n t heimulta neous extra ction of both E2m a n d H2m. Ten5-laser-shot MALDI -TOF spectra were ta ken fromach sample, with each spectrum taken from a dif fer-nt l ocat i on o n t he t a r g et . C a r e wa s t a k en d u r i ng d at acq u i s i t i o n t o mai nt ai n t he i o n s i g nal s i n t he u p p e r0–80%of the y -a xis ran ge a nd t o avoid driving indi-i d u al l as e r s ho t s i nt o s at u r at i o n. S p e ct r a we r e no r -

mal i z ed t o t he E 2m signal through baseline integra-ion , a n d t h e in tegr a l of H2m w a s d et er m in ed .

nt egra ls from the 10 spectr a t a ken for each calibra tiont a n d a r d w e r e a v e r a g e d a n d t h e s t a n d a r d d e v i a t i o n

wa s calcula ted. A ca librat ion curve wa s constructed byl o t t i ng t he av e r ag e o f t he no r mal i z e d i nt e g r al s f o rach s t a nd ar d v er s u s t he H2m concentr a tion.

Screening. U rine sam ples were collected from indi-iduals, treated with protease inhibitor cocktail , andooled to 4°C. The urine samples were centrifuged for 5

m i n ( a t 5 0 0 0g ) i mme d i at e l y p r i o r t o anal y s i s t o r e -mov e a ny p r eci pi t a t e d ma t e r ial . I n p r ep ar a t i on f orMSIA, 100 L o f e ach u r i ne s amp l e was mi x e d wi t h

100 L of stock h orse urine a nd 200 L of HBS. Thitrea tment is identica l to tha t used in prepara tion of thwo r ki ng cu r v e, wi t h t he e xce pt i on of r e pl aci ng t hs t a n d a r d w i t h t h e h u m a n u r i n e s a m p l e . M S I A w aperformed as described in the working curve section.

RESULTS AND DISCUSSION

M SI A T i p E val ua ti on/ B i oflui ds Screeni ng

The MSI A tips w ere evalua ted by screening a numb er of e as i ly ob t ai na b le b iofl u id s . The i nt ent of t hscreen wa s t o gauge the degree of nonspecific bindinencountered from each of the fluids and to briefly investigate alternative rinsing protocols that reduce contribut ions from n onspecifi c binding. F igure 2A shows MA LD I -T O F s p e ct r u m o f d i l u t e d hu man t e ar and s pe ct r u m s howi ng t e ar comp ou nd s r e t ai ne d d u r inMSIA. High-level proteins present in the tear s domnate the MALDI-TOF spectrum: lysozyme (MWcalc

14,696; MWobs 14,691) and tear lipocalin (MWcalc 17,444; MWobs 17,440). Other polypeptide signals aro b s e r v e d i n t he 2 - t o 5 -k D a r ang e , as we l l as a l o wintensity signa l at m /z 11,727 Da , presuma bly due t2m . T h e M S I A s p e c t r u m s h o w s s i g n a l s d u e t o t hselectively retained 2m (MWcalc 11,729; MWobs 11,731) a nd at t e nu at e d s ig nal s f or t he l y soz y me a not h e r n on s peci fi e d com pou n d s. F i gu r e 2B s h owM AL D I -TO F a n d M S I A s pect r a of d il ut e d h u m aplasma. As is commonly observed during direct analysis of serum or pla sma , t he MALDI-TOF spectrum idomina ted by signa ls origina ting from a lbumin. Othe

lower m /z s ignals are also present; however, 2m sign a l s a r e n ot ob ser v ed . Th e M S I A s p ect r u m s h owstrong signa ls due t o the selectively reta ined 2m a nfew other sign a ls from nonspecifi ed compounds. F igur2C shows spectra of diluted saliva (MALDI-TOF) ansalivary proteins reta ined during MS IA. The MALDITOF spectrum shows a number of signa ls in t he 1- t18-kDa ra nge, most prominent ly in t he peptide regionsignals corresponding to 2m ar e not ob s er v ed . ThMSIA spectrum, obta ined af ter using t he normal r insprotocols, shows signa ls due to t he selectively reta ine2m and an abundance of nonspecified compounds i

the low-molecular-ma ss ra nge. A second MS IA a na lys i s was p e r f o r me d i n whi ch an ad d i t i o nal r i ns e wi t0.05% (S D S ) wa s i ncl u de d b e t we en t he H B S and t hddH 2O rinses (Fig. 2C). The SDS rinse, al though nocompletely el iminating the low-mass signals, did significantly reduce their contribution to the mass spectrum without a proportional reduction of the 2m sign a l . F ig ur e 2D s h ow s s pect r a r es ult i ng fr om t ha na lysis of huma n urine. The MALDI -TOF spectrumshows a number of signals in the peptide region and aa bsence of signal for 2m. The MS IA spectrum is dom


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nat e d b y s i g nal s f r o m t he 2m , w i t h f e w a d d i t i o n a lignals from nonspecified compounds.

The porous glass fr i ts used in the MSIA tips were

udged to perform reasonably w ell in screening of t heiofl uids. Int ermediat e CMD a mplifi cation of the glass

ri ts provided a largely hydrophilic surface with mul-ple a tt a chment points (carboxylic a cid groups) for

oupling of the a nt ibody. As a result, th e an tibody loa df each t ip is more tha n suffi cient t o ca pture low levelsf 2m wi t ho u t s at u r at i o n o f t he ant i b o d y . A l s o , t heydr ophilic surfa ce can be wa shed free of most n onspe-i fi cal ly b ou nd comp ou nd s b y r i ns ing wi t h aq u eou sonic buffers. With the exception of the saliva sample,

MSIA exhibited reasonably clean mass spectra show-

ing predominantly signals derived from 2m. The SDwa sh of the sa l iva screen, a l though improving spectraq u al i t y , d i d no t co mp l e t e l y e l i mi nat e a l l o f t he no n

specified compounds. Upon closer investigat ion i t if ou nd t ha t t ho se comp ou nd s ( id ent i fi e d b y mas s alysozyme, -defensins, a nd hista tins) have p I s of 10suggesting retent ion via char ge intera ctions (w ith freecarboxyl groups) that are not broken by the moderatpH (7.8) and salt (150 mM NaCl) content of the HBbuffer. Thus, other r insing combinat ions (e.g. , highsa lt or different detergent s) w ill need to be investiga tei f t he s al i var y s cr e en i s d ee med t o b e o f b iol og icasignifi ca nce. I t is worth n oting, however, tha t t he presence of the nonspecifi ed compounds (in a ny of th e sa m

IG. 2. 2-Microglobulin MSIA screening of biofluids. Samples were prepared by dilution of the biofluid with HBS (H 2O for stand-alonM A L DI -TOF) a nd repet it ive-flow incuba t ion t hrough t he M SI A t ip. Tips w ere w a s hed us ing HB S a nd w a t er before elut ion of ret a ine

ompounds direct ly ont o a ma s s s pect romet er t a rget us ing -cyano-4-hydr oxycinnam ic a cid (satura ted in 1:2, a cetonitrile:H 2O; 0.2

ifluoroa cet ic a cid). (A) Huma n t ea rs . (B ) Huma n pla s ma . (C) Huma n s a liva —t he s a liva required a n a ddit iona l r ins e w it h 0.05%S DS (i

a ter) to reduce nonspecific binding. (D) Huma n urine. In a ll cases, 2m was efficiently retrieved from the biofluids using the flow-incubatnse procedure. The masses determined for the 2m (us ing ext erna l ca libra t ion) w ere w it hin 0.1% of the calculated value (MWcalc

1,729.7; MWt e a r s 11,735; MWplasma 11734; MWsaliva 11,742; MWurine 11,735).


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les) did not interfere w ith the una mbiguous determi-ation of 2m, w hich w a s identified by virtue of directetection a t i ts char a cteristic molecular ma ss.

Quantification

P rotein qua ntifi cation using MALDI -TOF requiress e o f i nt e r nal s t and ar d s t o co mp e ns at e f o r v ar y i ng

aser intensit ies a nd spot-to-spot differences in sa mple

omposition that give rise to fluctuations in analyte ionignal (18). Although proteins with characteristics un-ke those of the analyte may be used as internal stan-ar ds (a s ha s been shown during protein quan tifi ca tionirectly from mixtures (18, 19) or during MALDI-TOFua ntifi cation of a ffi nity-retrieved species by a dditionf an i nt e r nal r e f e r e nce s t and ar d t o p e p t i d e s e l u t e d

rom beaded affinity reagent (20)), internal referencet an d ar d s t h at b eha v e si mil ar l y t o t he ana l y t e du r i ngaser desorption/ionizat ion a re genera lly preferred.

This prerequisite is met during MSIA by choosing in-ernal references that share sequence homology with

h e ta rget protein: enzym a tic/chemica lly modified ver-ions of the targeted protein (15, 16, 21), truncated/xtended recombinant forms of the target proteins, thesame) target protein recombinantly expressed in iso-opica lly enriched medium (e.g., 15N or 18O ), or t hea me protein from a different biologica l species. Givenh a t t h e r e c e p t o r i s a b l e t o c a p t u r e b o t h t h e t a r g e tr o t e i n and t he i nt e r nal r e f e r e nce , MS I A can b e d e -igned a round a single receptor syst em (15, 21). Alter-at i ve ly , a t w o-r e ce pt o r s y s t em can b e cons i de r ed

where one receptor is used to retrieve the target pro-

t e in a n d a s ep a r a t e r ecep t or i s u s ed t o r e t r iev e t hinternal reference (15, 16, 22).

T h e i n t e r n a l r e f e r e n c e c h o s e n f o r t h i s s t u d y w aE2m , w h i ch s h a r e s 7 5% ho mo lo g y wi t h i t s hu macou n t e r pa r t a n d i s 300 D a low e r i n m a s s t h aH2m (t h u s , b ot h s p eci es s h a r e s i m i la r c h a r a c t er i st i c s a n d a r e e a s i l y r e s o l v e d i n t h e m a s s s p e c t r a )E v e n t h ou g h n o d a t a cou l d b e fo un d o n t h e r e la t i v

d i s s o c i a t i o n c o n s t a n t s b e t w e e n t h e p o l y c l o n a l a n t2m I g G a n d H2m o r E2m , p r el im i n a r y s t u d i es h ow e d t h a t t h e a n t i b od y e xh i b it e d c r os s -r e a c t i vi ts u f fi c ie n t t o r e t a i n b ot h s p ec ie s . F i g u r e 3 A s h ows p ect r a r e pr e s en t i n g M S I A a n a l y s es of H 2m s t a nd a r d s i n a con c en t r a t i on r a n g e of 0 .01 –1. 0 m g /LE a ch s p ect r u m , n o r m a l i ze d t o t h e E2m s i g n a l , i s of 10 65-la ser-shot spectra ta ken for ea ch ca libra tiopoin t . P l ot t i n g t h e a v er a g e of t h e 10 n or m a l iz eH2m i n t eg r a l s f or e a c h s t a n d a r d v er s u s t h e H 2mco nce nt r at i o n r e s u l t s i n t he wo r k i ng cu r v e s ho wn iF i g . 3 B . L i n e a r r e g r e s s i o n fi t t i n g o f t h e d a t a y i e l d

I H2m /I E2m 4.09 [H2m i n mg /L] 0.021 (R 2

0 .9 8 3 ), wi t h a wo r k i ng l i mi t o f d e t e ct i o n at a S /N 3 of 0.0025 mg/L (210 pM) a nd a l imit of qua nt ifi cation of 0.01 mg/L (850 pM). The st a nd a rd er ror of ap o i nt s o f t he wo r k i ng cu r v e i s 5%.

Quant itat ive Determinat ion of 2 m i n U r i n e S a m p l es

Ten sam ples were collected from four individualfemale (31 years, pregnant; one sample (F31)), mal(30 years; four samples over 2 days (M30)), male (3years; two samples over 2 days (M36)), and male (4

IG. 3. Qua nt it a t ive 2m-M SI A —w orking curve. (A) R epres ent a t ive s pect ra of da t a us ed t o genera t e t he w orking curve. Huma n 2oncentra tions of 0.01–1.0 mg/L were investigat ed. E quine 2m (MW 11, 396. 6) w a s us ed a s a n int erna l s t a nda rd. (B ) Working curv

enera t ed us ing t he da t a repres ent ed in (A). The t w o-deca de ra nge w a s s pa nned w it h good l inea rit y (R 2

0.983) and low standard erro

5%). Er ror bar s refl ect the st an dar d deviat ion of 10 repetitive 65-laser-shot spectra ta ken from each sa mple.


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e ar s ; t hr e e s amp l e s o v e r 2 d ay s (M4 4 )). A l l o f t he

n di vi du a l s w er e i n a s t a t e of g o od h ea l t h w h e n t h e

am ples w ere collected. R esults from MSIA of t he 10

rine samples are shown in Fig. 4. The bars depict the

2m concentration determined for each sample, while

he inset spectra above each bar show the respective

H2m signa ls norma lized to E2m. The da ta for t he 10

amp l e s s ho w r e mar k ab l e co ns i s t e ncy , wi t h an av e r -

ge 2m concentration of 0.100 0.021 mg /L (high ,.127 mg /L; low , 0.058 mg /L). An a dditiona l a na lysis

wa s p er f or me d o n a u r i ne s amp le o bt a i ned f r om an6-year-old female (F86) who had recently suffered a

e nal i nfe ct i on. B e cau s e of t he s ig ni fi cant l y hi g her

evel of 2m found in this sample (see inset spectrum)

wa s necessary t o qua nti ta tively dilute the urine by a

actor of 10 in order to keep the 2m s ig nal i ns id e t he

y n a m ic r a n g e of t h e w o rk in g cu r ve a n d a c cu r a t e ly

stablish the 2m concentr a tion in F 86 (a t 3.23 0.02

mg/L).

Posttr anslat ional M odi ficat ions

The mass-selective detection of MSIA makes possble the discovery an d qua ntifi cation of varia nts of 2mt h a t m a y b e p res en t in u rin e. D u r in g q u a n t it a t i vscreening of the urine samples, a second, higher-molecular-mass species (m 161 Da ) wa s coextra ctew i t h t h e 2m. The species is presumably a glycosylate(one h exose) form of 2m and is observed most promi

nently in F86. Figure 5 shows a n overlay of tw o MSIAspectra taken from the urine of F86 (diluted 20) anM36 (no dilution; given for comparison). The level oglycosylated 2m is much great er in F86 tha n in M36The specifi c ca use of th e elevat ed level of t he glyco-2mis at present uncertain.

P revious w ork ha s reported the qua ntifi cation of glycosylated H2m by direct MALDI-TOF analysis of serum into which glycosylated H2m w a s d ope d a t r easona bly high concentra tions (23). Due to poor m a sspectr a l resolution a nd int erferences from t he serum,

IG.4. Qua nt it a t ive 2m-MSIA—screening. H uma n urine sa mples from five ind ividuals w ere screened over a period of 2 da ys. The avera galue determin ed for healthy individuals (10 sam ples; four individua ls (three ma le; one female) ages 30– 44 year s) was 0.100 0.021 mg /L

he level determined for an 86-year-old female with a recent urinary tract infection indicated a significant increase in 2m concentratio

3.23 0.072 m g/L ).


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urve-fitting routine was used to deconvolute signalsrom mult iple H2m glycoforms, the integrals of which

were t hen n orma lized to w ild-type 2m for use in con-tr ucting a w orking curve of rea sonable linea rity (R 2 .8 8 ). A l t ho u g h t he d at a p r e s e nt e d he r e ar e s i g ni fi -

ant ly bett er an d clearly a ble to support rigorous qua n-fi ca tion without a id of fit t ing routines, i t is uncerta in

whether the working curve constructed for the unmod-fied 2m ca n be used directly for th e rigorous q uan ti-cation of the glycosylated 2m. Such correlation re-uires t ha t t he a ffi n it y con st a nt s for , a n d t heesorption/ioniza tion effi ciency of, both 2m f or ms a r equal—issues tha t w ere not investigat ed in t his study.

Re g ar d i ng t he af fi ni t y co ns t ant s , t he af fi ni t y -p u r i fi e dolyclonal antibody used in this study clearly showsroad cross-reactivity (as demonstrated by the coex-

r act i o n o f H2m a n d E2m) so i t is highly probable

h a t b o t h H2m f or ms a r e e xt r a ct e d w i t h s i mi lar e ffi -iencies. S imila rly , t he a ddition of a single ca rbohy-ra te moiety should not severely a tt enuat e the relativeesorption/ioniza tion efficiency. As a result, th e con-entra tion of t he glycosylat ed 2m form might be esti-

m a t e d u s i ng t h e 2m-w orking curve a t 0.072 mg/L—oughly the sa me concentr a tion as w ild-type 2m in therine sam ples from t he hea lthy individuals.

Regardless of whether both wild-type and glycosy-at e d 2m c a n b e q u a n t i fi e d u s i n g a s i n g l e w o r k i n gurve, it is importa nt to note tha t t he concentra tion of

the wild-type 2m determined during MSIA does accurately reflect the concentration of only the wild-typ2m and no t t he co mb i nat i o n o f b o t h o f t he s p e ci e sThu s , MS I A ho ld s a p ar t i cu l ar ad v a nt a g e o ve r o t het e chni q u es t ha t ar e u nab l e t o d i ff er e nt ia t e b e t we esimilar forms of a target analyte. In that elevated 2mlevels are used as a general indicator of immune system activity , while 2m-glycosylation has been assoc

a ted w ith more specific a i lments (e.g. , a dvan ced glycosylated end-products associated with dialysis relateamyloidosis (24)), MSIA is able to deconvolute thesindependent contributing factors and yield results thamor e accu r at e ly conne ct a s pe ci fi c b ioma r k er wi t h specific a i lment.

CONCLUSION

The experiments presented here were performed te val u at e t he u s e o f MS I A in t he a na l y si s of 2m. Thd e v e l o p me nt o f t he as s ay b e g an wi t h f as hi o ni ng d evices that facil i tated the rapid and efficient extractioof 2m from common biological fluids. Following on oup r e v i o u s wo r k whe r e p i p e t t o r t i p s we r e ad ap t e d f oaf fi ni t y e xt r a ct i on (15), MS I A t i ps we r e s pe ci fi cal lconstructed to have surface area sufficient for analytcaptur e w hile ma inta ining a low dea d (elution) volumeThe ma nufa cturing process used in th is report yieldet i ps w i t h a n es t im a t e d b in d in g ca p a c it y of 10 – 10pmol, w hile having a dead volume of a pproxima tely 1.L. This binding/elution ra tio wa s found to a dequa telm a t c h t h e 2m concentr a tions found in the biologica

fl uids. Moreover, t he effi cient ca pture of 2m using thtips kept t he sa mpling volume low (less tha n 100 L obiological fluid). In addition, the t ip chemistries employed during this study exhibited l it t le nonspecifibinding for three of the four biofluids and even in thf ou r t h (s al i va) d i d not i nt r od u ce an al y t i cal i nt er f erences into the analysis.

T he q u ant i t a t i v e cap ab i l i t i e s o f MS I A ar e cl e ar ldemonstra ted in t he w ork set forth in this report . Th2m con cen t r a t i on r a n g e i n ves t ig a t e d d u ri n g t h istudy (0.010–1.0 mg/L) is a dequa te to cover t he 2mlevels in all fluids. Good linearity is observed over th

t w o d ecad e r a ng e s (R 2 0.983) with a n overa ll erroof 5%. I mp or t a nt t o a ccu r at e q u ant i fi cat i on i s t hchoice of an appropriate reference standard, which it hi s e x amp l e was f u l fi l l e d b y u s e o f ho r s e u r i ne e nriched in 2m. H owever, even th ough th e horse urine iv ie we d a s an i d eal b ackg r ou nd me d iu m (b ecau s e mor e clos el y mi mi cs t he t r u e a nal y t i cal me di a t habuffers), i t wil l need t o be repla ced in future a na lysew i t h pu rifi e d E2m i n or d er t o e nsu r e cons i st e ncwhe n ana l y zi ng a l ar g e nu mb er of s a mp le s o ve r l onperiods of time.

IG. 5. M SI A s how ing eleva t ed level of glycos yla t ed 2m i n a n6-yea r-old fema le (da rk gra y). During M SI A , a s econd s igna l isbserved a t m 161 Da, indicating the presence of glycosylated

2m. MSIA is able to adequa tely resolve the tw o 2m forms, resultingn a more a ccura t e qua nt ifica t ion of t he na s cent 2m a nd pos s ibleuantification of the glycoprotein. Such differentiation is important

ons idering t ha t t he t w o 2m forms origina t e from (or a re ma rkersor) dif ferent a i lment s . M SI A of a hea lt hy individua l , s how ing l i t t le

lycosylat ion, is given for compar ison (light gra y).


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A l t ho u g h t he s cr e e ni ng s t u d y p r e s e nt e d he r e wasot extensive enough to be considered a clinical st udy, does demonstrate the uti l i ty of MSIA in accurately

dentifying and quantifying 2m present in urine sam-les. The four baseline individuals contributing urine

o the project were considered healthy, not sufferingr o m any k no wn g e ne t i c a f fli ct i o ns l i nk e d t o 2m ora ving suffered from a ny a i lments in th e month imme-

iat ely preceding t he a na lyses. Quali ta tive evalua tionf t he 2m retained from the samples revealed singleignals for 2m w ith m olecular ma ss corresponding t ohe wild-type sequence of the protein (within 0.02%xp er i ment a l e r r or ; s pe ct r a i nt er na l ly cal i br at e d b ysing the E2m signa ls; see Fig. 4). Quant ita tive ana l-sis within t he group show ed rema rkably consta nt 2mevels that are consistent with those found in controlroups during other st udies (25). B y cont ra st, t he urinea m p le ob t a i n ed f rom a n ol de r i n di vi du a l of poorealth showed a marked increase in urinary 2m level

30 times greater). I t should be noted that this esti-

m a t e w a s m a d e w i t h ou t i n t er fer en ce f r om a h i gh er -m a s s v a r i a n t of 2m, w hi ch wa s r e ad i l y d e t ect a b le i nhe ma s s s pe ct r a (s ee Fi g . 5 ). The mos t r e as onab l exp lan at i on f or t he ob s er v at i on of t w o ma s s -s hi ft e d

yet relat ed) signals in t he ma ss spectrum is the pres-nce of : (i ) t he wi l d -t y p e p r ot e in and (i i) a v ar i antxi st i ng d u e t o e i t he r a g ene t ic p ol y mor p his m or aosttranslational modification. In this particular case,

he var iant is most easily identifi ed by the ma ss shif t of161 Da a s a glycosylat ed form of 2m; v ar i ant s d u e t o

enetic polymorphisms a re essentia l ly ruled out be-ause the ma ss shift is grea ter tha n tha t resulting from

ny single nucleotide polymorphism (i .e. , [TG G ] G G G ]; r e su l t ing i n Tr p G l y ; d m 129.15 Da ).

H o we v e r , had v ar i ant s p o s s e s s i ng a s i g ni fi cant mas shif t (15 Da) as a result of a genetic polymorphismeen present in the sample, they would have been aseadily recognized as the glycosylated 2m.

Fi nal l y , i n t hat MS I A anal y s e s ar e f a i r l y r ap i d ande l at i v e l y e as y t o p e r f o r m, t he ap p r o ach l e nd s i t s e l farticularly well to the rapid development of analytical

methods and the a na lysis of la rge numbers of sam ples.The rapid rate of analysis opens the possibility of real-

me method development in which changes in incuba-

on and rinse protocols can be readily implementedwhe n t he r e s u lt s f r om a ju s t comp le t ed t r i a l a r e an a-yzed. Once developed and optimized, these methodsould be readily applied to screening of biological flu-ds. During this study samples were analyzed at a ratef ap p r ox ima t e ly t hr e e p e r hou r , whi ch al l owe d f oreveral analyses to be performed on a given individual

w ith in a single da y (as show n in Fig. 4, M30 on 10/01).This ra te of ana lysis w a s essentia l ly l imited by instru-m en t a t ion t h a t is n ot d es ig ned t o a ccom m od a t emultiple sam ples—each a na lysis in t his study (prepa-

ration-through-analysis) was performed individuallyHowever, i t is fea sible to increase t his ra te of an a lysito hundreds-per-day by use of parallel pipetting sta

t i on s a n d m a s s s pe ct r om et e rs t h a t a c cep t m u lt i pls amp l es o n a n a r r ay e d f or mat . I n t hi s ma nne r , pi pet i ng s t a t i ons ad d r e s si ng 96-we l l f or mat t i t e r p lat ema y be used to simultan eously incubat e an d rinse thmultiple sa mples a nd th en elute onto a ma ss spectrom

e t e r t a r g e t o f t h e s a m e f o r m a t . G i v e n t h e a b i l i t y ti ncr e as e s amp l e t hr o u g hp u t , MS I A s t and s t o fi nd i ncreased use in large-sca le cl inica l a pplications wh ere xce pt i onal q u al i t a t i ve a nd q u ant i t a t i ve a ccu r acy a rboth needed in protein a na lysis.

ACKNOWLEDGMENTS

This publicat ion wa s supported in pa rt by G ra nt 1 R43 GM5660

01A2 from t he Na t iona l I ns t i t ut es of H ea lt h. I t s cont ent s a re s ole

the responsibility of the aut hors and do not necessarily represent th

offi cia l v iew s of t he Na t iona l I ns t i t ut es of Hea lt h.

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